G_αq sensitizes TRPM8 to inhibition by PI(4,5)P₂ depletion upon receptor activation

ABSTRACT

Activation of G-protein coupled receptors (GPCRs) was proposed to inhibit the cold and menthol sensitive Transient Receptor Potential Melastatin 8 (TRPM8) channels via direct binding of G_αq to the channel. It is well documented that TRPM8 requires the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂ or PIP₂] for activity. It was claimed however that a decrease in cellular levels of this lipid does not contribute to channel inhibition upon receptor activation. Here we show that supplementing the whole cell patch pipette with PI(4,5)P₂ reduced inhibition of TRPM8 by activation of G_αq-coupled receptors in mouse dorsal root ganglion (DRG) neurons. Activation of the same receptors induced Phospholipase C (PLC) activation and decreased plasma membrane PI(4,5)P₂ levels in these neurons. PI(4,5)P₂ also reduced inhibition of TRPM8 by activation of heterologously expressed G_αq-coupled muscarinic M1 receptors. Co-expression of a constitutively active G_αq protein that does not couple to PLC inhibited TRPM8 activity, and in cells expressing this protein decreasing PI(4,5)P₂ levels using a voltage sensitive 5’-phosphatase induced a stronger inhibition of TRPM8 activity than in control cells. Our data indicate that PI(4,5)P₂ depletion plays an important role in TRPM8 inhibition upon GPCR activation, and G_αq inhibits the channel by reducing its apparent affinity for PI(4,5)P₂ and thus sensitizes the channel to inhibition by decreasing PI(4,5)P₂ levels.